Animal Reproduction Science 130 (2012) 33–41

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Pregnancy-specific protein B (PSPB), progesterone and some

biochemical attributes concentrations in the fetal fluids and serum and
its relationship with fetal and placental characteristics of Iraqi riverine
buffalo (Bubalus bubalis)
T.A. Abdulkareem a,∗ , S.M. Eidan a , M.A. Ishak a , S.A.M. Al-Sharifi b , M.A. Alnimer c ,
C.W. Passavant d , J.R. Branen d , R.G. Sasser d
a
Department of Animal Resources, College of Agriculture, University of Baghdad, Baghdad, Iraq
b
Ministry of Agriculture, Baghdad, Iraq
c
Department of Animal Production, Faculty of Agriculture, University of Jordan, Ammin, Jordan
d
BioTracking LLC, Moscow, ID, USA

a r t i c l e i n f o a b s t r a c t

Article history: This study was carried out to demonstrate the pregnancy-specific protein B (PSPB), proges-
Received 4 November 2011 terone and some biochemical parameters concentrations in amniotic fluid, allantoic fluid
Received in revised form 12 January 2012 and fetal serum collected from slaughtered Iraqi riverine pregnant buffaloes at three dif-
Accepted 14 January 2012
ferent months of gestation (6th, 7th and 8th). Ten out of 22 adult buffaloes of 4.6 ± 0.97
Available online 23 January 2012
years old were used in this study. The buffaloes were mated naturally by monitoring the
estrus cycles via appearance of vaginal fluids and mounting by bulls. Pregnancy was che-
Keywords:
PSPB
cked for these buffaloes by non-returning to estrus for three estrus cycles and assured by
Progesterone rectal palpation on day 61 post-mating (PM). Buffaloes were slaughtered at three different
Biochemical attributes periods of gestation (three at 6th month, four at 7th month and three at 8th month of ges-
Blood tation) to verify the progesterone and PSPB as well as some blood attributes levels (glucose,
Fetal fluids cholesterol, total protein, albumin, globulins and albumin: globulins ratio) in amniotic fluid
Buffaloes (AF), allantoic fluid (LF) and fetal serum (FS). Progesterone was higher (P < 0.01) in LF at the
8th month of gestation and lower in FS during the 7th and 8th months of pregnancy. PSPB
concentrations were greater in FS (6th and 8th months in particular) than in both AF and LF.
The overall mean of cholesterol concentration was higher in FS (P < 0.05) followed by AF and
LF that had the lowest concentration. The FS exhibited higher total protein during the three
gestation periods. Most of fetal and placental measurements increased as the pregnancy
advanced. In conclusion, these results described, for the first time, the PSPB and progeste-
rone concentrations and blood characteristics in fetal fluids and serum in water riverine
buffaloes during different stages of pregnancy. Progesterone concentrations were greater
in allantoic fluid than in other fluids. In contrast, PSPB and other blood attributes were
higher in fetal serum than other fluids of Iraqi riverine buffaloes. These findings reflect the
changes in hormones, proteins and other metabolites during different gestation periods.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Amniotic fluid is crucial to fetal health because it forms

a protective sac around the fetus that prevents mecha-
∗ Corresponding author. Tel.: +964 7801697514. nical and thermal shock, possesses antimicrobial activity,
E-mail address: talal200320032000@yahoo.com (T.A. Abdulkareem). assists in acid/base balance, and contains nutritional

0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2012.01.002
34 T.A. Abdulkareem et al. / Animal Reproduction Science 130 (2012) 33–41
factors (Stewart et al., 2001). A wide range of proteins has
been identified in amniotic fluid (Drohse et al., 1998). These
proteins can enter the amniotic fluid from the maternal
uterine tissues, umbilical cord, amniotic fluid cells, fetal
urine, and other fetal secretions that include transuda-
tion through fetal skin (Jauniaux et al., 1998). Amniotic
fluid proteins are principally of maternal origin and have
concentrations lower than in maternal serum (Jauniaux
et al., 1994). Allantoic fluid is composed of hypotonic urine,
maintains osmotic pressure of the fetal plasma and pre-
vents fluid loss to maternal circulation (Jainudeen and
Hafez, 2000).
Placental aspartic acid proteinases were first discove-
red by Butler et al. (1982) when they reported the finding of
pregnancy-specific protein B (PSPB). The PSPB, by immuno-
histochemical methods, was found in the binucleated cells
of the ruminant trophectoderm (Eckblad et al., 1985; Sasser
and Ruder, 1987). This was repeated by Zoli et al. (1992). A
radioimmunoassay (RIA) was developed for measurement
of PSPB and was used to find the protein in the circulation
of cattle (Sasser et al., 1986). This was the first establish-
ment of a protein marker for pregnancy in cattle. The PSPB
is a protein fraction containing several proteins not a single
protein (Sasser et al., 1987, 1989). Plasma PSPB/pregnancy-
associated glycoprotein (PAG) measurements have been
used to characterize the embryonic mortality in cattle
(Humblot et al., 1988; Gábor et al., 2007; Lopez-Gatius
et al., 2007; Eidan, 2008). Recently, PSPB have been also
used to detect the early pregnancy in Iraqi water buffalo
(Abdulkareem et al., 2011). The bovine PSPB fraction was
characterized as glycoproteins showing relative molecular
mass (Mr) between 47 and 53 kDa (Butler et al., 1982) fol-
lowed by a report of other molecular masses up to 80 kDa
with different isoelectric points (pI) from 4.0 to 4.4 (Sasser
et al., 1989). The pI’s were closely related to the amounts
of sialic acid of each isoform. Nine years after the work of
Butler et al. (1982) and Zoli et al. (1991), using the methods
of Butler et al. (1982), and with testing of isolates by Sas-
ser’s laboratory, isolated an aspartic proteinase and gave it
the name of pregnancy-associated glycoprotein designated
as PAG-1-67 because of its Mr was 67 kDa. Four isoforms
(pI: 4.4, 4.6, 5.2 and 5.4) were detected in the initial pre-
paration (Beckers et al., 1999). The nucleotide sequences
coding for a form of PSPB (Lynch et al., 1992) and PAG-
1 (Xie et al., 1991) showed that PAG-1 was homologous
to one of the variants (Sasser et al., 1987, 1989) of PSPB
as reviewed by Sasser et al. (2009) and are in the aspartic
acid protease family of proteins. The nucleotide sequence
of related molecules of aspartic acid proteases of the pla-
centa has been identified and some of these proteins are
also released into maternal circulation where they can be
assayed by different RIA methods (Sousa et al., 2001). A
family of PSPB molecules was identified from the placenta
of ewes by Atkinson et al. (SBU3; 1993) and Willard et al.
(PSPB; 1995). The SBU3 glycoproteins, with Mr of 69, 62 and
57 kDa, showed partial amino acid homology to the bovine
forms of PSPB and PAG1. The PSPB of the elk and moose
also showed various immunoreactive forms (Huang et al.,
1999). Pregnancy-specific protein B or PSPB, the first pla-
cental aspartic proteases to be discovered and named, is
the name that will be used in this paper.
The enzyme-linked immunosorbent assay (ELISA) mea-
sures the presence of PSPB in the blood of cattle and buffalo.
Accuracy is an essential consideration for this type of tech-
nology. Careful evaluation of the assay for PSPB showed
that if a cow was detected as not-pregnant the test was cor-
rect over 99% of the time. If it detected a cow as pregnant, it
was correct 91–94% of the time. This lesser percentage was
partially due to loss of embryos before a follow-up test was
conducted. Detection of pregnancy by radioimmunoassay
for PSPB has been done in several ruminant animals inclu-
ding deer (Wood et al., 1986), mountain goat (Houston
et al., 1986), muskoxen (Rowell et al., 1989), sheep (Willard
et al., 1995) goats (Humblot et al., 1990), elk (Haigh et al.,
1993; Huang et al., 2000) moose (Haigh et al., 1993; Huang
et al., 2000) and water buffalo (Abdulkareem et al., 2011).
The measurements of PSPB using ELISA test, progeste-
rone and other biochemical characteristics in fetal fluids
and serum during different pregnancy stages have not
previously studied. Therefore, this study was designed to
demonstrate the concentrations of PSPB, progesterone and
some biochemical attributes in fetal fluids and serum of
Iraqi riverine buffaloes.
2. Materials and methods
2.1. Animals
Ten pregnant females out of 22 mated riverine buffa-
loes of 6.0 ± 0.93 years of age were employed in this study.
Animals were naturally mated following estrus detected
via monitors both day and night. Pregnancy was che-
cked for these buffaloes by non-returning to estrus for
three estrus cycles and assured by rectal palpation on day
61 post-mating (PM). The pregnant buffaloes were selec-
ted randomly from the whole pregnant females (n = 14).
Buffaloes were housed at Al-Thahab Al-Abiath village, Abu-
Ghraib, Baghdad during the period from August 2005 to
November 2007. Buffalo were fed green roughages (alfalfa
and a barley: clover mixture) ad libitum in addition to
4 kg/head/day of concentrate consisting of barley grains;
35%, wheat bran; 30%, maize; 17%, cotton; 10%, soybean
meal; 5% as well as salt, vitamins and minerals; 3%. Ani-
mals were vaccinated against brucellosis, foot and mouth
disease, hemorrhagic septicemia and rinderpest.
2.2. Experimental design
Buffaloes were slaughtered at three different periods of
gestation (three at 6th month, four at 7th month and three
at 8th month of gestation) to measure the progesterone and
PSPB as well as some of biochemical attributes concentra-
tions (glucose, cholesterol, total protein, albumin, globulins
and albumin: globulins ratio) in fetal fluids. Following
slaughtering, the uterus was removed. Ten milliliters of
amniotic, allantoic fluids and fetal serum were collected.
The uterus was opened carefully. The entire feto-placental
unit was completely separated from the uterus. The fetus
was weighted and the crown rump (the length between
occipital condyle of head till the base of the tail) was recor-
ded (Rexroad et al., 1974). The blood sample (10 ml) was
collected via heparinized vacutainer tubes were collected
 T.A. Abdulkareem et al. / Animal Reproduction Science 130 (2012) 33–41
from each fetus. Plasma was immediately harvested from
blood following centrifugation of the sample (1409 g for
15 min.) and was stored at −20 ◦ C until assay. Placentomes
were separated from the uterine wall and embryonic mem-
branes and the total weight and numbers were recorded.
Length and diameter of nine placentomes were estima-
ted using a cloth tape and vernier caliper respectively
and mean values were calculated. These placentomes were
taken randomly from different sites of the placenta, nine
cotyledons (fetal side) were carefully separated from the
caruncles (maternal side) and the weight of paraplacental
tissues and cotyledons: caruncles were also determined.
2.3. Progesterone assay
Radioimmunoassay was used to measure the plasma
progesterone concentration (ng/ml). A commercial kit was
used (Immunotech & Beckman Coulter Ltd., Marseille,
France). The assay was carried out at the Laboratory
of Animal Reproductive Physiology, College of Agricul-
ture, University of Jordan. The analytical sensitivity of the
assay was 0.05 ng/ml; whereas, intra-assay and inter-assay
coefficients of variation were 5.8% and 9% respectively.
The cross-reactivity (specificity) was 100%, 20.18%, 7.31%,
2.38% and 0.22% with progesterone, 5␤-Pregnanedione,
5␣-pregnanedione, 6␤-hydroxyprogesterone and 17␣-
hydroxyprogesterone respectively. The cut-off value of
progesterone level used to detect pregnant buffalo cows
was 1 ng/ml.
2.4. PSPB quantitative assay
Plasma PSPB was assayed quantitatively using the pro-
cedure provided by BioTracking LLC, Moscow, ID, USA and
as modified and validated for Iraqi buffalo (Abdulkareem
et al., 2011).
2.5. Biochemical aspects determination in fetal fluids and
serum
Glucose (Cooper, 1973) and cholesterol (Allain et al.,
1974) concentrations were quantitatively determined
using the kit provided by Teco diagnostic Company, Ana-
heim, CA, USA. Total protein was assessed using Biuret’s
method (Green et al., 1982). Bush (1998) was employed
for quantitative determination of albumin concentration.
The kit was provided by Kudna Medical Company, Amman,
Jordan. A Globulins concentration was calculated by sub-
tracting plasma albumin from plasma total protein (Otto
et al., 2000; Zvorc et al., 2000). Albumin to globulins ratio
was estimated dividing albumin concentration by globulins
concentration (Zvorc et al., 2000).
2.6. Statistical analyses
Statistical computations were performed using general
linear model (GLM) procedure in the SAS program (SAS,
2001) to investigate the effect of gestation, type of fluid and
their interaction on PSPB, progesterone, blood biochemical
35
parameters as well as fetal and placental characteristics.
The statistical model for analysis of variance was:
Yijk =  + Fi + Oj + FOij + eijk
where
Yijk = dependent variable (progesterone and PSPB concen-
trations, glucose, cholesterol, total protein, albumin,
globulins, albumin: globulins ratio and fetal and placental
characteristics).
 = Overall mean.
Fi = effect of fluid type (F = amniotic, allantoic and fetal
serum).
Oj = effect of gestation month (O = 6th, 7th and 8th).
FOij = effect of interaction between fluid type × gestation
month.
eijk = Error term.
Differences among means were computed using the
Duncan multiple range test (Duncan, 1955).
3. Results
3.1. Progesterone concentrations in fetal fluids and serum
Generally, low levels of progesterone were detected
in amniotic fluids (AF) of Iraqi riverine buffaloes. The
concentrations did not change over the three months of
gestation studied and were 0.07 ± 0.03, 0.11 ± 0.03 and
0.09 ± 0.04 ng/ml for the 6th, 7th and 8th months of ges-
tation, respectively (Table 1).
Differences in progesterone concentrations in allantoic
fluids (LF) during the experimental periods were similar
(P > 0.05). However, a 4-fold increase in progesterone
levels was noted during the 8th month (0.50 ± 0.43 ng/ml)
in comparison to 6th (0.11 ± 0.07 ng/ml) and 7th
(0.12 ± 0.03 ng/ml) months (Table 2).
Non-significant variations were detected in fetal serum
(FS) progesterone concentrations during the entire three
months of gestation. They were 0.07 ± 0.04, 0.02 ± 0.01 and
0.03 ± 0.01 ng/ml for the 6th, 7th and 8th months respecti-
vely (Table 3).
3.2. PSPB concentrations in fetal fluids and serum
The PSPB concentrations in AF were three times
higher (P < 0.01) during the 6th month of gestation
(7.03 ± 2.49 ng/ml) in relation to value at the 7th month
of gestation (2.14 ± 0.13 ng/ml; Table 3). Differences in ng
PSPB/ml were not appreciable between 6th and 8th months
of pregnancy in spite of the 2-fold increase in the former
one as compared with the latter (Table 3).
Significant differences were not seen in ng PSPB/ml
in LF over the study period. However, there was a ten-
dency toward an increase of PSPB levels during the
8th month (4.38 ± 3.29 ng/ml) in comparison to the 6th
(+40%, 2.60 ± 0.76 ng/ml) and 7th (+7%, 1.23 ± 0.35 ng/ml)
months of gestation (Table 2). Concomitantly with
LF pattern, ng PSPB/ml in FS did not differ signifi-
cantly across gestation months. It was numerically
36 T.A. Abdulkareem et al. / Animal Reproduction Science 130 (2012) 33–41

Table 1
PSPB, progesterone concentrations and some biochemical characteristics of amniotic fluids in Iraqi riverine buffaloes during different gestation periods
(mean ± S.E).

Trait Month of gestation

6th 7th 8th

Progesterone (ng/ml) 0.07 ± 0.03a 0.11 ± 0.03a 0.09 ± 0.04a

PSPB (ng/ml) 7.03 ± 2.49a 2.14 ± 0.13b 3.14 ± 0.89ab
Glucose (mg/dl) 67.77 ± 12.32a 63.88 ± 2.05a 60.95 ± 2.44a
Cholesterol (mg/dl) 138.55 ± 3.92a 138.70 ± 4.60a 121.09 ± 21.04a
Total protein (g/100 ml) 7.48 ± 0.14a 7.51 ± 0.08a 7.66 ± 0.19a
Albumin (g/100 ml) 2.30 ± 0.14a 2.47 ± 0.14a 2.34 ± 0.16a
Globulins (g/100 ml) 5.19 ± 0.24a 5.04 ± 0.12a 5.33 ± 0.03a
Albumin:globulins ratio 0.45 ± 0.05a 0.49 ± 0.04a 0.44 ± 0.03a

Means with different superscripts within each row differ significantly (P < 0.05).

Table 2
PSPB, progesterone concentrations and some biochemical characteristics of allantoic fluids in Iraqi riverine buffaloes during different gestation periods
(mean ± S.E).

Trait Month of gestation

6th 7th 8th

Progesterone (ng/ml) 0.11 ± 0.07a 0.12 ± 0.03a 0.50 ± 0.43a

PSPB (ng/ml) 2.60 ± 0.76a 1.23 ± 0.35a 4.38 ± 3.29a
Glucose (mg/dl) 60.88 ± 1.61a 66.48 ± 2.08a 61.01 ± 3.02a
Cholesterol (mg/dl) 115.13 ± 23.53a 145.67 ± 6.58a 110.64 ± 22.86a
Total protein (g/100 ml) 7.54 ± 0.12a 7.40 ± 0.02a 7.61 ± 0.13a
Albumin (g/100 ml) 2.42 ± 0.21a 2.68 ± 0.17a 3.04 ± 0.25a
Globulins (g/100 ml) 5.11 ± 0.25a 4.72 ± 0.18a 4.56 ± 0.25a
Albumin:globulins ratio 0.48 ± 0.06a 0.58 ± 0.06a 0.68 ± 0.09a

Means with similar superscripts within each row did not differ significantly (P > 0.05).

higher at the 6th month (11.30 ± 1.00 ng/ml) compared
to the 7th (−30.9%, 7.81 ± 1.23 ng/ml) and 8th (−12%,
9.94 ± 0.17 ng/ml) months of pregnancy (Table 3).
3.3. Biochemical parameters in fetal fluids and serum
Mean AF glucose concentrations did not change signi-
ficantly across the pregnancy periods (Table 1) in spite of
an 8.23% increase in LF glucose concentration during the
7th month of gestation (66.48 ± 2.08 mg/dl) (Table 2). The
overall mean of glucose FS did not change during the study
period. However, it tended to be greater (+26.8%) at the 6th
month of gestation (82.83 ± 15.22 mg/dl) as compared with
8th month of pregnancy (60.63 ± 15.34 mg/dl) (Table 3).
Cholesterol concentration in AF did not change during
the three months of pregnancy, but, numerically it ten-
ded to be lower during 8th month (121.09 ± 21.04 mg/dl)
as compared with the 6th (138.55 ± 3.92) and the 7th
(138.70 ± 4.60 mg/dl) months of gestation (Table 1). The
LF cholesterol concentrations during the three months of
pregnancy were not significantly different, being generally
higher during the 7th month (145.67 ± 6.58 mg/dl) and
lower at the 8th (110.64 ± 22.86 mg/dl; Table 2). Results
of cholesterol levels in FS resembled those of AF and LF
patterns, where the differences across months lacked signi-
ficance (Table 3).
Non-significant differences were noted in total
protein concentrations across the three gestation
periods in AF, LF and FS fluids. It ranged from
7.48 ± 0.14 to 7.66 ± 0.19 g/100 ml (AF), 7.40 ± 0.02
to 7.61 ± 0.13 g/100 ml (LF) and 9.19 ± 0.40 to
9.57 ± 0.73 g/100 ml (FS) (Tables 1–3).
Albumin concentrations in AF appeared to be simi-
lar across the pregnancy periods, namely 2.30 ± 0.14,

Table 3
PSPB, progesterone concentrations and some biochemical characteristics of fetal serum in Iraqi riverine buffaloes during different gestation periods
(mean ± S.E).

Trait Month of gestation

6th 7th 8th

Progesterone (ng/ml) 0.07 ± 0.04a 0.02 ± 0.01a 0.03 ± 0.01a

PSPB (ng/ml) 11.30 ± 1.00a 7.81 ± 1.23ab 9.94 ± 0.17a
Glucose (mg/dl) 82.83 ± 15.22a 73.17 ± 17.05a 60.63 ± 15.34a
Cholesterol (mg/dl) 148.58 ± 20.19a 142.91 ± 13.97a 148.15 ± 14.51a
Total protein (g/100 ml) 9.35 ± 0.08a 9.19 ± 0.40a 9.57 ± 0.73a
Albumin (g/100 ml) 3.21 ± 0.10b 3.97 ± 0.34a 3.05 ± 0.15b
Globulins (g/100 ml) 6.14 ± 0.15a 5.21 ± 0.70a 6.52 ± 0.88a
Albumin:globulins ratio 0.52 ± 0.03b 0.83 ± 0.17a 0.50 ± 0.10b

Means with different superscripts within each row differ significantly (P < 0.05).
 T.A. Abdulkareem et al. / Animal Reproduction Science 130 (2012) 33–41
2.47 ± 0.14 and 2.34 ± 0.16 g/100 ml for the 6th, 7th and
8th months of pregnancy, respectively (Table 1). Coinci-
ding with the AF trend, mean albumin concentrations in
LF were not different during the study periods. The ave-
rage ranged from 2.42 ± 0.21 to 3.04 ± 0.25 g/100 ml, which
approximates to the values in AF (Table 2). The trend of
FS albumin was slightly different from both AF and LF.
A greater (P < 0.01) mean value of FS albumin was found
in the 7th (3.97 ± 0.34 g/100 ml) as compared with the
6th (3.21 ± 0.10 g/100 ml) and 8th (3.05 ± 0.15 g/100 ml)
months of pregnancy (Table 3).
The mean values of AF globulins concentrations
during the study periods were showed in Table 1.
Non-appreciable variations occurred in globulins concen-
trations across the months studied. However, there
was an increasing trend during the 8th month of pre-
gnancy (5.33 ± 0.03 g/100 ml) in relation to the two other
months. Similar pattern of globulins concentrations were
seen in LF results. The average was non-significantly
higher at the 6th (5.11 ± 0.25 g/100 ml) and lower during
the 8th (4.56 ± 0.25 g/100 ml) as compared to the 7th
(4.72 ± 0.18 g/100 ml) months of gestation (Table 2).
Results pertaining to FS globulins did not reveal signifi-
cance across the study periods. However, it tended to be
higher at the 8th month (6.52 ± 0.88 g/100 ml) than either
at the 6th (+5.8%) or the 7th (+21%) months of pregnancy
(Table 3).
Results of albumin to globulins ratio indicated that
differences across months either in AF or in LF lacked
significance (Tables 1 and 2). Higher (P < 0.01) albumin to
globulins ratio of FS appeared during the 7th month of
pregnancy (0.83 ± 0.17) as compared to the 6th (+37.3%,
0.52 ± 0.03) and 8th (+41%, 0.50 ± 0.10) months of gestation
(Table 3).
3.4. Fetal and placental characteristics during three
months of gestation
As pregnancy advanced, the fetal weight was 6.6
and 2.5 folds heavier (P < 0.05) at the 8th month
(13.73 ± 4.68 kg) than at the 6th (2.07 ± 0.54 kg) and
the 7th (5.51 ± 2.01 kg) months of pregnancy respecti-
vely (Table 4). The crown rump was highly significantly
different (P < 0.01) by 42.6 cm and 22% at the 8th
month of gestation (57.50 ± 3.62 cm) relatively to the 6th
(33.00 ± 3.00 cm) and the 7th (44.75 ± 4.61 cm) months of
gestation respectively (Table 4). There were no significant
differences in the total number of placentomes across the
three months studied. Mean numbers were 133.67 ± 12.35,
208.25 ± 25. 27 and 161.00 ± 22.74 for the 6th, 7th and
8th months of gestation (Table 4). There were higher but
non-significant increases in the total weight of cotyle-
dons per buffalo female at the 8th month of pregnancy
(1140.00 ± 120.14 g) in relation to the weight during the
6th (570.00 ± 173.88 g) and the 7th (760.00 ± 214.90 g)
months. These increases were 1.5 and 2 times, respecti-
vely (Table 4). The length and diameter of placentomes
tended to increase as the pregnancy advanced. They were
higher in the 8th month of gestation as compared to the
two other months studied (Table 4). The average weight
of individual placentome was greater (P < 0.05) during the
37
8th month of pregnancy (29.82 ± 7.20 g) as compared to
the 6th (10.83 ± 0.85 g) but it was not greater than the
average weight during the 7th (15.95 ± 4.66 g) month of
gestation (Table 4). Cotyledon weight per buffalo female
tended to be 50% and 36.7% heavier at the 8th month of
gestation (12.04 ± 4.83 g) compared to the weight at the 6th
(6.04 ± 0.68 g) and 7th (7.62 ± 2.23 g) months, respectively
(Table 4). The caruncle weight per buffalo was signifi-
cantly (P < 0.02) greater as the pregnancy advanced (8th
month, 16.85 ± 3.33 g; Table 4). The cotyledon: caruncle
ratio was lower (P < 0.01) during the 8th month of gestation
(0.69 ± 0.19) in relation to the ratio at the 6th (1.45 ± 0.12)
month. Nevertheless, it was not significantly different from
that at the 7th (1.07 ± 0.09) month of pregnancy (Table 4).
The average weight of paraplacental tissues did not change
significantly as the pregnancy advanced. However, it wei-
ghed slightly less at the 6th month of gestation compared
to the two other months (Table 4).
3.5. Correlations among progesterone, PSPB and fetal
and placental characteristics
Correlation coefficient showed positive correlation
between PSPB and fetus weight (0.64) and length of placen-
tome (0.65). The relationships between PSPB and other fetal
and placental traits studied lacked significance (Table 5).
All fetal and placental traits studied were not significantly
correlated with fetal progesterone concentration (Table 5).
Interestingly, fetus weight had a highly significant posi-
tive correlation with average weight of caruncle (0.88),
cotyledon (0.93) placentome (0.97), and total weight of
cotyledons (0.77) and the length (0.81) and diameter (0.91)
of placentome, as well as with crown rump length (0.90)
(Table 5). Similarly, the crown rump had a highly posi-
tive relationship with average weight of caruncle (0.90)
and cotyledon (0.73), weight of placentome (0.89), length
(0.75) and diameter (0.84) of placentome as well as with
total weight of cotyledons (0.83) (Table 5). There were also
a significant association between total weight of cotyle-
dons and average weight of caruncle (0.84) and cotyledon
(0.72), weight of placentome (0.85) as well as length (0.81)
and diameter (0.72) of placentome (Table 5). Length of pla-
centome was positively correlated with average weight of
caruncle (0.84) and cotyledon (0.82), weight of placentome
(0.88) and diameter of placentome (0.85) (Table 5).
4. Discussion
This study describes for the first time the progeste-
rone and PSPB concentrations in buffalo fetal fluids and
serum. Generally, low progesterone concentrations were
detected in amniotic and allantoic as well as in fetal serum
during the three months that were studied. Progesterone
is secreted directly from the corpus luteum and placenta
to the maternal circulation (Bearden et al., 2004). A very
low non-significant correlation coefficient between plasma
progesterone and fetus weight (0.002) was noted in the
present study and may confirm this notion. To our know-
ledge, no other previous study dealt with the PSPB levels in
buffalo’s embryonic fluids and fetal serum. The PSPB pre-
paration (Fraction R-37) used for development of the ELISA
38 T.A. Abdulkareem et al. / Animal Reproduction Science 130 (2012) 33–41

Table 4
Fetal and placental characteristics of Iraqi riverine buffaloes during three months of gestation (mean ± S.E).

Trait Gestation month

6th 7th 8th

Fetus weight (kg) 2.07 ± 0.54b 5.51 ± 2.01ab 13.73 ± 4.68a

Crown rump (cm) 33.00 ± 3.00b 44.75 ± 4.61ab 57.50 ± 3.62a
No. of placentomes 133.67 ± 12.35a 208.25 ± 25.27a 161.00 ± 22.74a
Total weight of cotyledons (g) 570.00 ± 173.88a 760.00 ± 214.90a 1140.00 ± 120.14a
Length of placentome (cm) 4.39 ± 0.51a 5.00 ± 0.50a 5.94 ± 0.68a
Diameter of placentome (mm) 12.76 ± 0.17a 15.11 ± 3.21a 22.84 ± 4.93a
Average weight of placentome (g) 10.83 ± 0.85b 15.95 ± 4.66ab 29.82 ± 7.20a
Average weight of cotyledons (g) 6.04 ± 0.68a 7.62 ± 2.23a 12.04 ± 4.83a
Average weight of caruncles (g) 4.16 ± 0.35b 7.35 ± 2.27b 16.85 ± 3.33a
Cotyledon:caruncle ratio 1.45 ± 0.12a 1.07 ± 0.09ab 0.69 ± 0.19b
Average weight of para-placental tissues (g) 0.62 ± 0.29a 0.98 ± 0.30a 0.94 ± 0.24a

Means with different superscripts within each row differ significantly (P < 0.05).

(Howard et al., 2007) consists of several protein molecules
of various molecular weights and isoelectric points that
show cross reactivity to PSPB derived antibodies (Sasser
et al., 1989). The relationship between PSPB and PAG’s
has previously been discussed (Sasser et al., 2009). The
PSPB fraction and the term PSPB should not be conside-
red as a single molecule but as a protein fraction and, thus,
is not directly comparable to PAG-1. The protein fraction
contains PAG-1 based on the finding that a specific mole-
cular clone of PSPB identified in the fraction was identical
to a molecular clone named PAG-1. This result is due to the
cross-recognition of PAG-1 by some PSPB derived antibo-
dies. The cloning of additional PAG’s has likely lead to the
specific molecular identification of several other proteins
in the original PSPB fraction (Sasser et al., 1989). The RIA
(Sasser et al., 1986) and subsequent ELISA (Howard et al.,
2007) assays using antibodies developed against the PSPB
fraction (Fraction R-37; Sasser et al., 1989), detect antigens
in the serum of pregnant cows from as early as 15 days
of pregnancy until the postpartum period (Sasser et al.,
1986). Based on the published timing of the expression of
the PAG genes (Green et al., 2000) it seems highly probable
that the profile of response in the PSPB assays is due to
recognition of a varying combination and concentration of
proteins (including PAG’s) expressed throughout gestation.
If the PSPB assays were highly specific to only one aspartic
protease, such as PAG-1, the wide range of effectiveness
early, and across gestation, would not have been obtai-
ned. The appreciable PSPB concentrations were found in
embryonic fluids and fetal serum with the highest concen-
tration during the 6th month of pregnancy. This coincides
with high maternal PSPB concentrations during similar
pregnancy period and decline at term (Abdulkareem et al.,
unpublished data). Increased fetal and placental weights
during the second trimester of gestation may explain these
highest levels of PSPB. Additionally, the high positively
correlation between PSPB levels and fetus weight (0.64)
found in the present study may strengthen this postula-
tion. The high level of glucose concentrations in fetal serum
in relative to amniotic and allantoic fluids reveal glucose
as the major metabolic fuel for the fetus and are transfer-
red across the placenta by means of an active transport
system (Jainudeen and Hafez, 2000). However, the fetal
blood glucose levels are lower than those of the mother
(approximately 30% in calf and 25% in lambs) (Comline and
Silver, 1974). In all three fluids, there are non-significance
differences in glucose concentrations indicating stability of
this nutrient throughout pregnancy as the major source of
energy for fetus. It reflects maternal nourishment stability
qualitatively and quantitatively. Increasing maternal feed
intake in late pregnancy resulted in about 20% increase in
fetal glucose concentrations in the absence of any changes
in fetal plasma leptin concentrations (Mühlhäusler et al.,
2002). For the three fluid types, total protein concentra-
tions were remarkably unchanged, though it appeared to
be greater during the 8th month of gestation in relation
to other months. This seems to be common as the pre-
gnancy advances in order to meet the fetus requirements.
Fetal serum exhibited higher total protein and its fractions
levels (albumin and globulins) as compared with those
of AF and LF. This is due to the important role of pro-
teins in regulation of growth and differentiation (Jainudeen
and Hafez, 2000). However, the amniotic fluid proteins are
regarded as nutritional status indicators of normal fetal
growth (Tisi et al., 2004). The placenta is a multifaceted
organ that plays critical roles in maintaining and protec-
ting the developing fetus (Wilson, 2002). Heavier fetuses
with greater crown rump were found during the 8th month
in relation to the two other months. This may be attri-
buted to higher trophectoderm proliferation during the
last months of gestation in relative to the others. Higher
and faster proliferations of the trophectoderm were noti-
ced during the last trimester of pregnancy as compared
with earlier gestation periods to meet the faster growth
of the fetus during this period in pigs (Wilson and Ford,
1997). A larger variation and higher bPSPB production
were observed in cows carrying crossbred fetuses derived
from IVF techniques than in purebred inseminated cows,
suggesting that there was a relationship between fetal
growth rate and PSPB increase during pregnancy in cattle
(Vasquez et al., 1995). Increasing trophectoderm prolife-
ration is consistent with increasing placental weight and
size (Rivera et al., 1997). Supporting this observation, grea-
ter placental weights and measurements were observed
during the 8th month in relation to the 6th and 7th months.
Variations in placental vascularity contribute to the varia-
tions in placental size and functions as pregnancy advances
(Wilson, 2002). The highly significant positive correlation
Table 5
Correlation coefficients among PSPB, progesterone and some fetal and placental characters of Iraqi riverine buffaloes during different gestation periods.

Average weight Cotyledon: Average Average Average Diameter of Length of Total Number of Crown Fetus PSPB Progesterone

T.A. Abdulkareem et al. / Animal Reproduction Science 130 (2012) 33–41

of para-placental caruncle ratio weight of weight of weight of placentome placentome weight of placentomes rump weight
tissues caruncle cotyledon placentome cotyledon

Average weight of –
para-placental
tissues
Cotyledon:caruncle −0.13 –
ratio
Average weight of 0.11 −0.64 –
caruncles
Average weight of 0.11 0.10 0.77** –
cotyledons
**
Average weight of 0.16 −0.42 0.95 0.93** –
placentome
Diameter of 0.12 −0.40 0.94** 0.90** 0.97** –
placentome
Length of placentome 0.02 −0.32 0.84** 0.82** 0.88** 0.85** –
Total weight of 0.26 0.41 0.84** 0.72** 0.85** 0.81** 0.72** –
cotyledons
Number of 0.45 −0.03 0.004 0.20 0.11 0.009 0.31 0.12 –
placentomes
Crown rump 0.44 0.60 0.90** 0.73** 0.89** 0.84** 0.75** 0.83** 0.32 –
Fetus weight 0.20 −0.37 0.88** 0.93** 0.97** 0.91** 0.81** 0.77** 0.19 0.90** –
PSPB 0.03 −0.03 0.54 0.58 0.59 0.50 0.65* 0.45 0.12 0.52 0.64* –
Progesterone 0.101 −0.58 0.105 −0.15 −0.005 −0.04 0.05 −0.000 0.39 0.21 0.002 −0.46 –
*
P < 0.05.
**
P < 0.01.

39
40 T.A. Abdulkareem et al. / Animal Reproduction Science 130 (2012) 33–41
(0.65) between PSPB concentration and placentomal length
confirmed the hypothesis that increasing PSPB secretions
form placental binucleated cells with enlargement of pla-
cental measurements and decreased with the placenta at
parturition. Wooding (1992) revealed that trophectoderm
binucleated cells disappear at the end of pregnancy and just
before parturition in cattle. This would explain the loss of
PSPB at that time. Concomitantly, Klisch et al. (2006) found
that terminal N-acetyl-galactosamine (detected by Dolichos
biflorus agglutinin, DBA) in placentomal binucleated cells
is greatly reduced prior to parturition in cattle, confirming
the disappearance of these cells prior to parturition. Strong
immunostaining PSPB spots were observed exclusively in
the cytoplasm of large binucleated cells found predomi-
nantly in fetal cotyledonary tissues (Eckblad et al., 1985).
Our data demonstrate for the first time that progeste-
rone concentrations were greater in allantoic fluid than in
other fluids of Iraqi riverine buffaloes. In contrast, PSPB and
other blood attributes were higher in fetal serum than other
fluids, and these may reflect the changes of these metabo-
lites during different gestation period.
Acknowledgments
The authors are grateful to Dr. M.J. Tabaa in the
Department of Animal Production, Faculty of Agriculture,
University of Jordan for calculation assistance. The tech-
nical assistance of Mr. A. Swaid (Animal Reproductive
Physiology Lab.) and Miss Samar (Virology Lab.) in carrying
out this study is also acknowledged.
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